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Antibodies 2019, 8(1), 10; https://doi.org/10.3390/antib8010010

A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells

1
Pharmaceutical Biotechnology, Eberhard Karls University, 72076 Tuebingen, Germany
2
Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany
*
Author to whom correspondence should be addressed.
Received: 4 December 2018 / Revised: 4 January 2019 / Accepted: 7 January 2019 / Published: 10 January 2019
(This article belongs to the Special Issue Nanobody)
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Abstract

Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens. View Full-Text
Keywords: nanobodies; chromobodies; live-cell imaging; compound screening; cellular models nanobodies; chromobodies; live-cell imaging; compound screening; cellular models
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Keller, B.-M.; Maier, J.; Weldle, M.; Segan, S.; Traenkle, B.; Rothbauer, U. A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells. Antibodies 2019, 8, 10.

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